We will use our computational design methodology, based on an explicit physical model of protein-DNA interfaces, to design novel homing endonuclease variants predicted to cleave specifically within sites in XSCID and other therapeutically important genes. Genes corresponding to the designed proteins will be synthesized, the in vitro and in vivo cleavage specificities determined in Component 2 - Monnat, Component 4 - Scharenberg, and Component 5 - Stoddard groups, improved variants obtained using molecular evolution by Component 4 - Scharenberg, and structures determined of promising designs in Component 5 - Stoddard. These data will be used to refine and improve our computational design methodology. Shortcomings of the physical model underlying our current approach include the limited treatments of backbone flexibility and of water-mediated hydrogen bonding interactions which can contribute significantly to the energetics of protein-DNA interactions, and we will use the experimental feedback to improve both aspects of our model; for example the crystal structures will guide our approach to modeling backbone flexibility. We will use the improved computational design methods to design a second round of endonucleases with therapeutically important cleavage specificities and these will be characterized as in the first round and the experimental feedback used to further refine the computational method. By cycling in this way between detailed computational modeling and in depth experimental characterization we aim to develop robust methods for creating tailored enzymatic reagents for targeted genetic therapies via gene correction.